The hydroxyphenylpyruvate dioxygenases are enzymes which catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. This reaction takes place in the presence of iron and in the presence of oxygen (Crouch, N. P. et al., Tetrahedron, 53, 20, 6993-7010, 1997). It may be hypothesized that the HPPDs contain an active site which is capable of catalyzing this reaction, in which iron, the substrate and the molecule of oxygen link together.
Some molecules which inhibit this enzyme, and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate, are also known. Some of these molecules have been used as herbicides since inhibition of the reaction in plants leads to whitening of the leaves of the treated plants and to the death of the said plants (Pallett, K. E. et al. 1997 Pestic. Sci. 50 83-84). The herbicides for which HPPD is the target, and which are described in the state of the art, are, in particular, isoxazoles (EP418175, EP470856, EP487352, EP527036, EP560482, EP682659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, which is a selective herbicide for maize, diketonitriles (EP496630, EP496631), in particular 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3 phenyl)propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3Cl2phenyl)propane-1,3-dione, triketones (EP625505, EP625508, U.S. Pat. No. 5,506,195), in particular sulcotrione, or else pyrazolinates.
In addition, the amino-acid sequence of hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P.J. 874 has been described (Ruetschi et al.: Eur. J. Biochem. 205, 459-466, 1992). At U.S. Pat. No. 6,268,549, a sequence of a gene of this type is described and that such a gene can, once incorporated into plant cells, produce an over-expression or an activation of HPPD in the plants giving to the latter an worthwhile tolerance to certain novel herbicides, such as those of the isoxazoles family or that of the triketones. The sequence can be of bacterial origin, such as especially the genus Pseudomonas or alternatively of plant origin, such as especially of monocotyledonous or dicotyledonous plants, especially of Arabidopsis or of Umbelliferae, such as, for example, the carrot (Daucus carotta). It can be isolated native or wild or possibly mutated artificially while at the same time fundamentally retaining a property of herbicidal tolerance against HPPD inhibitors, such as herbicides of the isoxazoles family or that of the triketones.
Also described at U.S. Pat. No. 6,245,968 is a mutant of such HPPD sequences. It is useful in the process of over expressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant sufficient to have enough functional enzyme in spite of the presence of the inhibitor. The patent describes that by mutating the enzyme in the vicinity of its C-terminal part, it was possible to obtain functional (enzymatically active) HPPDs which were less sensitive to HPPD inhibitors, such that expression of these functional HPPDs in plants improves the tolerance of the plants to HPPD inhibitors. Mutants which are enzymatically active or functional retain a significant portion of HPPD catalytic activity, and in the case of plants transformed with the sequences, the mutated sequences should preferably retain sufficient HPPD activity to sustain the growth of the plant.
With this mutation an amino acid of the primary sequence is replaced with another amino acid. By aligning these known sequences, by using the customary means of the art, such as, for example, the method described by Thompson, J. D. et al, (CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22; 4673-4680, 1994), and accessing these computer programs for sequence alignment which include a wide variety of tools, and are accessible via the Internet, for example, the skilled person is able to define the sequence homologies in relation to a reference sequence and find the key amino acids or else define common regions, making it possible, for example to align the sequence and locate a referenced position and to define a C-terminal region and an N-terminal region on the basis of this reference sequence.
In the case of the present invention, the reference sequence is the Pseudomonas sequence, with all the definitions and indications of the positions of particular amino acids being made with respect to the primary Pseudomonas HPPD sequence, here SEQ ID NO: 4 (this is the sequence identified as sequence 31 of the '968 patent). That reference sequence is shown in FIG. 1 of the '968 patent and depicts an alignment of several HPPD sequences which are described in the state of the art; these sequences are aligned with respect to the Pseudomonas HPPD sequence as the reference sequence and comprise the HPPD sequences as described therein of Streptomyces avermitilis (Genebank SAV 11864), of Daucus carota (Genebank DCU87257), of Arabidopsis thaliana (Genebank AF047834), of Zea mays (Genbank NM001112312), of Hordeum vulgare (Genebank HVAJ693), of Mycosphaerella graminicola (Genebank AF038152), of Coccicoides immitis (Genebank COITRP) and of Mus musculus (Genebank MU54HD). This figure gives the numbering of the amino acids of the Pseudomonas sequence and also the amino acids which are common to these sequences, with these amino acids being designated by an asterisk. On the basis of such an alignment, it is straightforward, from the definition of the Pseudomonas amino acid by its position and its nature, to identify the position of the corresponding amino acid in another HPPD sequence (with the alignment of sequences of different plant, mammalian and bacterial origin demonstrating that this method of alignment, which is well known to a skilled person, can be generalized to any other sequence). An alignment of different HPPD sequences is also described at Maxwell et al. US Patent Publication 20030066102 and Patent Application WO97/49816. The C-terminal part of the HPPDs, which is where the active site of the enzyme is located, differs from its N-terminal part by a linking peptide which ensures the stability of the enzyme and its oligomerization (the Pseudomonas HPPD is a tetramer while the plant HPPDs are dimers). The linking peptide makes it possible to define the N-terminal end of the C-terminal part of the enzyme, with the said peptide being located between amino acids 145 and 157 in the case of Pseudomonas. The C-terminal part can therefore be defined as consisting of the sequence defined, on the one hand, by the linking peptide and, on the other hand, by the C-terminal end of the enzyme, with the mutation which is effected in the C-terminal part of the HPPD therefore being effected in the region which has thus been defined. Two amino acids, which are in positions 161 and 162 in the case of the Pseudomonas sequence (D=Asp161 and H=His162), will be noted in all sequences. With reference to the Pseudomonas HPPD, it is therefore possible to define the linking peptide which represents the N-terminal end of the C-terminal part of the HPPD as being located between approximately 5 and 15 amino acids upstream of the amino acid Asp161. The mutation of interest in the present invention is effected on amino acids being located with reference to the Pseudomonas sequence at position 336.
There is a need to identify antibodies that are immunoreactive with the mutant HPPD protein described above so that plants containing such mutated HPPD protein can be readily identified. Especially useful would be an antibody that immunoreacts with Pseudomonas HPPD and especially the mutant HPPD protein containing the mutation at residue 336 (glycine (gly or G) to tryptophan (trp or W)). A method that would avoid time-consuming lab steps would reduce costs, allowing for quick identification of the transgenic plants containing the mutant protein, aiding in breeding and selection. Furthermore, antibodies that are immunoreactive with such proteins could be useful in isolating and purifying the proteins.
All references cited are incorporated herein by reference. The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 22, 2009, is named 210004.txt, and is 12,549 bytes in size.